1. Field of the Invention
The present invention is in the field of medical instrumentation. More specifically it relates to a small and portable sensor capable of making non-invasive measurements of average glucose levels and the aging process over time by quantifying fluorescent chemical molecular species in blood and tissue.
2. The Prior Art
An estimated 14 million Americans have diabetes mellitus with comparable prevalence rates in the rest of the world. The recently completed Diabetes Control and Complications Trial sponsored by the National Institutes of Health has confirmed that good glucose control over time can influence positively several outcome variables in this disease including blindness and kidney failure (Report: Diabetes Control and Complications Trial (DCCT) Summary and Recommendations reported at the 53rd Annual Meeting of the American Diabetes Association Jun. 12-15, 1993 Las Vegas, Nevada). Thus providing useful measures of glucose control and creating programs that enable persons with diabetes to control the illness have taken on even greater importance.
At the present there are two general methods by which glucose control can be quantified over time: self blood glucose monitoring by the individual subject and measurement of glycosylated proteins. Both of these approaches require a sample of blood. Home blood glucose monitoring provides information regarding the blood glucose at the moment and allows immediate correction of metabolic problems. Measurement of glycosylated or glycated proteins allows the quantification of glucose control over a longer period of time such that the overall efficacy of a management strategy can be determined. The DCCT utilized both approaches.
Measurement of glycated proteins has primarily used hemoglobin because it is easily obtained and because there is no new synthesis of hemoglobin in the red cell after three days in the circulation. Therefore whatever glycation occurs must be the result of a postsynthetic (post-translational) reaction and reflect the metabolic environment to which the hemoglobin has been exposed. The 120 day lifespan of the red cell allows the retrospective quantification of average glucose levels over time. The art of separating minor hemoglobins will be reviewed in some detail since it pertains to the present invention. In addition, the same chemical process has been shown to occur with other proteins in the body and proceed to the generation of fluorescent species known as advanced Maillard reaction products or nonenzymatic browning products or melanoidins which comprise the subject of measurement of the present invention.
The documentation that there is nonenzymatic post translational modification of proteins by sugars (glycation) has provided clinicians with a measurement of glucose control and investigators with a testable hypothesis regarding the biochemical basis for a number of pathological consequences of hyperglycemia. While the latter remains an hypothesis, the former has become fixed in the clinical equipment and methods used especially in medicine.
Studies in the clinical literature prior to the advent of measurements of glycated proteins remain difficult to interpret and controversial, in part because of the confounding influence of lack of documentation of "glucose control". Claims of "good control" versus "bad control" unencumbered by objective evidence produced more heat than light in the field of diabetes until the clinical availability of glycosylated or glycated hemoglobin assays.
Just as it is difficult to interpret clinical studies in diabetes without evidence of "control" as reflected by a well performed assay of glycation, so it is difficult to judge the level of clinical care in a given patient without such a measurement performed at intervals which allow evaluation of ongoing glycemia. Any assessment of care for patients with diabetes has to include glycemia as reflected by such an assay. The goal of modern diabetes management has become reestablishment of the normal internal milieu while avoiding hypoglycemia. Therefore, every clinician needs to be familiar with the glycosylation assay provided by his/her laboratory and be able to interpret the results in depth to the patient.
2.1 Separation of hemoglobin variants
The study of hemoglobin variants has provided a number of significant medical insights. In 1949, Linus Pauling and coworkers analyzed hemolysates by moving boundary electrophoresis and found that the hemoglobin from a patient with sickle cell anemia had a different mobility from that of a normal individual. This experiment provided the first identification of a "molecular disease". Pauling L, Itano H, Singer SJ, and Wells IC: Sickle cell anemia: A molecular disease. Science 1949; 110:543-4.
In normal human erythrocytes, hemoglobin A (Hb A or A.sub.0) comprises about 90 percent of the total hemoglobin. Besides Hb A, human red cells contain other hemoglobin components that are of considerable interest. Some of these, such as Hbs A.sub.2 and F, like sickle hemoglobin (Hb S) are products of alternate globin chain genes, and others such as HbA.sub.1c are post-translational modifications of Hb A.
In 1955, Kunkel and Wallenius analyzed human hemolysates by starch gel electrophoresis and found a minor component that had less negative charge than HbA and comprised about 2.5% of the total. This component was designated Hb A2 with two alpha and two a chains and was found to be elevated in individuals with 3 thalassemia. Kunkel HG and Wallensius G: New hemoglobins in normal adult blood. Science 1955; 122:228-9. Kunkel HG, Ceppellini R, Muller-Eberhard U, Wolf J: Observations on the minor basic hemoglobin components in the blood of normal individuals and patients with thalassemia. J Clin Invest 1961:36:1615-21. They also noted several hemoglobin species which had more negative charge, one of which was undoubtably Hb A.sub.1c. As early as 1958, Allen and coworkers noted that human hemoglobin could be separated into at least 3 minor components using column chromatography. Allen DW, Schroeder WA, Balog J: Observations on the chromatographic heterogeneity of normal adult and fetal human hemoglobin. J Am Chem Soc 1958:80:1628-34. The minor components were labelled in order of their elution from the column; hence, hemoglobins A.sub.1a, A.sub.1b, A.sub.1c.
During the 1960's it was demonstrated that a hexose molecule attaches to the hemoglobin structure in the fast eluting components. Holmquist WR, Schroeder WA: A new N-terminal blocking group involving a Schiff base in hemoglobin A.sub.1c. Biochemistry 1966;5: 2489-2503. Bookchin RM, Gallop PM: Structure of hemoglobin Alc: Nature of the N-terminal beta chain blocking group. Biochim Biophys Res Commun 1968;32:86-93. In 1962 Huismann and Dozy using gel electrophoresis found an increased level of minor hemoglobin components in a few diabetic patients treated with tolbutamide. Huisman THJ, Dozy AM: Studies on the heterogeneity of hemoglobin. V. Binding of hemoglobin with oxidized glutathione. J. Lab Clin Med 1962;60: 302-19. However, this finding remained unheeded until Rahbar, also using gel electrophoresis, re-discovered the existence of an elevation in these minor fractions in two patients with diabetes mellitus and later confirmed the observation in 140 diabetic patients. Rahbar S: An abnormal hemoglobin in red cells of diabetics. Clin Chem Acta 1968; 22:296-8. Rahbar S, Blumenfeld O, Ranney HM: Studies of an unusual hemoglobin in patients with diabetes mellitus. Biochem Biophys Res Commun 1969; 36:838-43. Trivelli et al (using the column cation exchange chromatographic method which became standard) found that in persons with diabetes mellitus, the concentration of Hb A.sub.1c was 2-3 times higher than in non-diabetic individuals. Trivelli LA, Ranney HM, Lai H-T: Hemoglobin components in patients with diabetes mellitus. N Eng J Med 1971; 248:353-7. In 1975 Tattersall et al studied a series of monozygotic twins of whom only one had diabetes and found elevated Hb A.sub.1c levels in the diabetic twins only. Because of a low correlation with fasting plasma glucose levels, they concluded that the measurement did not correlate with glycemia per se. Tatersall RB, Pyke DA, Ranney HM, Bruckheimer SM: Hemoglobin components in diabetes mellitus: Studies in identical twins. N Engl J Med 1975; 293: 117103.
In 1976, it became clear that Hb A.sub.1c resulted from a post translational modification of hemoglobin A by glucose and that there was a clinical relationship between Hb A.sub.1c and fasting plasma glucose, peak on the glucose tolerance test, area under curve of the glucose tolerance test, and mean glucose levels over the preceding weeks. Bunn HF, Haney Dn, Kamin S, Gabbay KH, Gallop PM. The biosynthesis of human hemoglobin A.sub.1c. J Clin Invest-57:1652-9. Koenig RJ, Peterson CM, Kilo C, Cerami A, Williamson JR: Hemoglobin A.sub.1c as an indicator of the degree of glucose intolerance in diabetes. Diabetes 1976; 25:230-232. Koenig RJ, Peterson CM, Jones RL, Saudek CD, Lehrman M, Cerami A: Correlation of glucose regulation and hemoglobin A.sub.1c in diabetes mellitus. N Engl J Med 1976; 295:417-20. It soon became apparent that an improvement in ambient blood glucose levels resulted in correction (inter alia) of Hb A.sub.1c levels (Peterson CM, Jones RL, Koenig RJ, Melvin ET, Lehrman ML: Reversible hematologic sequelae of diabetes mellitus. Ann Int Med 1977; 86:425-429. Peterson CM, Koenig RK, Jones RL, Saudek CD, Cerami A: Correlation of serum triglyceride levels and hemoglobin A.sub.1c concentrations in diabetes mellitus. Diabetes 1977; 26:507-9) and that these nonenzymatic glycosylation reactions might provide an hypothesis which could explain a number of the pathological sequelae of diabetes mellitus via toxicity arising from glucose adduct formation with proteins or nucleic acids. Peterson CM, Jones RL: Minor hemoglobins, diabetic "control" and diseases of postsynthetic protein modification. Ann Int Med 1977; 87: 489-91.
2.2 Chemistry of glucose adduct formation
As early as 1912, Maillard suggested that the chemical reactions that now bear his name might play a role in the pathology associated with diabetes mellitus. Maillard LC: Reaction generale des acides amines sur les sucres; ses consequences biologiques. C.R. Acad Sci 1912; 154:66-68. The ability of reducing sugars to react with the amino groups of proteins is now widely recognized as is the natural occurrence of many nonenzymatically glycosylated proteins. Important details as to the nature of such reactions are, however, still unclear.
The initial step (or Early Maillard Reaction) involves the condensation of an amino moiety with the aldehyde form of a particular sugar. Only a very small fraction of most common sugars is normally present in the aldehyde form (Table 1). Angyal SJ: The composition of reducing sugars in solution, in Harmon RE (ed): Asyummetry in Carbohydrates. New York, Marcel Dekker, 1979, pp 15-30. Benkovic SJ: Anomeric specificity of carbohydrate utilizing enzymes. Methods Enzymol 1979; 63:370-9. A number of transformations are possible following the addition of an amine to a sugar carbonyl group. Considerable evidence now exists which supports the involvement of an Amadori-type rearrangement for the adduct of glucose with the N-terminus of the B-chain of hemoglobin. The labile Schiff base aidimine adduct is transformed to a relatively stable ketoamine adduct via the Amadori Rearrangement.
TABLE 1 ______________________________________ % OF SUGARS IN ALDEHYDE FORM ______________________________________ Glucose 0.001 Ribose 0.017 Fructose 0.25 Glucose 6-P 0.4 Fructose 6-P 4-5 ______________________________________
Since hemoglobin circulates in its red cell for approximately 120 days, there is some opportunity in this cell for Late Maillard Reactions or enzymatic browning to occur. In these late Maillard Reactions, the Amadori product is degraded into deoxyglucosones which react again with free amino groups to form chromophores, fluorophores, and protein cross links. Hayase F, Nagaraj RH, Miyata S, Njoroge FG, Monnier VM: Aging of proteins: immunological detection of a glucose-derived pyrrole formed during Maillard Reaction in vivo. J Biol Chem 1989; 263:3758-3764. Peterson CM (ed): Proceedings of a conference on Nonenzymatic Glycosylation and Browning Reactions: Their Relevance to Diabetes, Mellitus. Diabetes 1982; 31 (Suppl 3) 1-82. In tissues which are longer lived, these reactions may be important mediators of diabetes pathology as well as the aging process. Although the structure of a large number of nonenzymatic browning products has been elucidated, few have been obtained under physiological conditions, thus making detection in vivo difficult and their pathological role uncertain. Horiuchi S, Shiga M, Araki N, Takata K, Saitch M, Morino Y: Evidence against in vivo presence of 2-(2-furoyl)-4(B)-(2-furanyl)1H-imidazole, a major fluorescent advanced end product generated by nonenzymatic glycosylation. J Biol Chem 1988;263:18821-6.
Table 2 summarizes the hypotheses whereby Maillard Reactions might contribute to the multiple pathologies associated with hyperglycemia. Peterson CM, Formby B: Glycosylated proteins, in Alberti KGMM, Krall LP (eds): Diabetes Annual 1 , New York, Elsevier, 1985, pp 1781 97. Peterson CM, Formby B: Glycosylated Proteins, in Alberti KGMM Krall LP (eds): Diabetes Annual 2. New York, Elsevier, 1986, pp 137-155. Kowluru RA, Heidorn DB, Edmondson SP, Bitensky MW, Kowluru A, Downer NW, Whaley TW, Trewhella J: Glycation of calmodulin: Chemistry and structural and functional consequences. Biochem. 1989; 28:2220-8. Arai K, Maguchi S, Fujii S, Ishibashi H, Oikawa K, Taniguchi N: Glycation and inactivation of human Cu-Zn-superoxide dismutase. Identification of the in vitro glycated sites. J Biol Chem 1987;262: 16969-78. Kaneshige H: Nonenzymatic glycosylation of serum IgG and its effect on antibody activity in patients with diabetes mellitus. Diabetes 1987; 36:822-8. In addition to the numerous studies of Early Maillard Reactions, the Late Maillard Reactions have been shown to increase concomitantly with diabetes related pathologies. Brownlee M, Cerami A, Vlassara H: Advanced products of nonenzymatic glycosylation and the pathogenesis of diabetic vascular disease. Diabetes Metab Rev. 1988; 4: 437-51. Monnier VM, Sell DR, Abdul Karim FW, Emancipator SN: Collagen browning and cross-linking are increased in chronic experimental hyperglycemia. Relevance to diabetes and aging. Diabetes 37:86772. Cohen MP: Diabetes and Protein Glycosylation: Measurement and Biologic Relevance. New York, Springer-Verlag, 1986. McCance DR, Dyer DB, Dunn JA, Bailie KE, Thorpe SR, Baynes JW, Lyons TJ. Maillard reaction products and their relation to complications in insulin-dependent diabetes mellitus. J. Clin. Invest. 91: 2470-2478, 1993. It remains difficult to establish whether these glycosylation changes are indeed causal of pathology associated with diabetes mellitus. J. Clin. Invest. 91: 2470-2478, 1993.
TABLE 2 ______________________________________ Hypotheses regarding the potential role of non-enzymatic glucosylation and browning in the pathology associated with diabetes mellitus ______________________________________ I Structural proteins A Collagen: Decreased turnover, flexibility, solubility; increased aggregating potential for platelets, binding of immunoglobulins, crosslinking, and immunogenicity B Lens crystallins and membrane: opacification, increased vulnerability to oxidative stress C Basement membrane: increased permeability, decreased turnover, increased thickness D Extracellular matrix: changes in binding to other proteins E Hemoglobin: change in oxygen binding F Fibrin: decreased enzymatic degradation G Red cell membrane: increased rigidity H Tubulin: cell structure and transport I Myelin: altered structure and immunologic recognition II Carrier proteins A Lipoproteins: alternate degradative pathways and metabolism by macrophages and endothelial cells, increased immunogenicity B Albumin: alteration in binding properties for drugs and in handling by the kidney C Ig G: Altered binding III Enzyme systems A Cu--Zn Superoxide Dismutace B Fibrinogen: altered coagulation C Antithrombin III: hypercoagulable state D Purine nucleoside phosphorylase: aging of erythrocytes E Alcohol dehydrogenase: substrate metabolism F Ribonuclease A: lose of activity G Cathepsin B: loss of activity H N-acetyl-D-glucosaminidase: loss of activity I Calmodulin: decreased calcium binding IV Nucleic acids Age-related changes, congenital malformations V Potentiation of other diseases of post-synthetic protein modification A Carbamylation-associated pathology in uremia B Steroid cataract formation C Acetaldehyde-induced changes in alcoholism ______________________________________
2.3 Terminology, pros, and cons of various measurements
Table 3 summarizes terminology used for hemoglobin which has been reacted with sugars.
TABLE 3 ______________________________________ Hemoglobin (Hb) Terminology ______________________________________ "Fast" Hemoglobin. The total HbA.sub.1 fractions (HbA.sub.1a, HbA.sub.1a2, HbA.sub.1b, HbA.sub.1c) which, because of more negative charge, migrates toward the anode on electrophoresis and elutes earlier on cation exchange chromatography than HbA.sub.c. Fetal Hemoglobin (HbF). The major hemoglobin component of newborn blood. HbF co-elutes with HbA.sub.1c by column chroma- tography. Glucosylated Hemoglobin. Hemoglobin modified by glucose at beta chain valine residues and epsilon amino groups of lysine residues. Glycated Hemoglobin. A term favored by biochemists to indicate adducts of sugars and hemoglobin which are formed non- enzymatically. Glycosylated Hemoglobin (glyco-hemoglobin). A generic term for hemoglobin containing glucose and/or other carbohydrate at either valine or lysine residues thus the sum of glycosyl adducts. Hemoglobin A. The major adult form of hemoglobin. A tetramer consisting of two alpha and two beta chains (alpha.sub.2, beta.sub.2). Hemoglobin A.sub.c. The major component of HbA identified by its chromatographic and electrophoretic properties. Post-translational modifications, including glycosylation do exist, but do not significantly affect the charged properties of the protein. Hemoglobin A.sub.1. Post-translationally modified, more negatively charged forms of HbA.sub.c (primarily glycosylation at the beta chain terminal valine residue) separable from HbA.sub.c by chromatographic and electrophoretic methods. Hemoglobin A.sub.1a1, HbA.sub.1a2, HbA.sub.1b, HbA.sub.1c. Chromatographically distinct stable components of HbA.sub.1. Hemoglobin A1.sub.1a1, HbA.sub.1a2, HbA.sub.1b : "Fastest" most anionic forms of HbA consisting primarily of adducts of phosphorylated glycoyl- tic intermediates with HbA.sub.c. Hemoglobin A1.sub.c. Component of HbA.sub.1 which consists of 50 to 90% hemoglobin (depending on the quality of resolution of the chromatographic system) glucosylated by a ketamine linkage at the beta chain terminal valine residue. Pre-Hemglobin A.sub.1c. A labile form of glycosylated Hb containing glucose bound in aldimin linkage to the beta chain terminal valine residue. Hemoglobin-AGE. Advanced Maillard or glycosylation end pro- ducts bound to hemoglobin. Circulate in the red cell and corre- lates with the amount of hemoglobin A.sub.1c. ______________________________________
HbA.sub.1c is one of several minor hemoglobins, but because of its relatively high concentration in normal persons (3% to 6% of normal hemoglobin), it is the one most extensively studied. Because circulating red blood cells or erythrocytes are incapable of initiating protein synthesis, HbA.sub.1c is produced as a post-synthetic modification of hemoglobin A.sub.0 (or adult hemoglobin). The rate of modification depends on the mean circulating sugar (glucose) levels to which the erythrocyte is exposed. The post-synthetic modification of hemoglobin A to form HbA.sub.1c is nearly irreversible, and its rate of synthesis reflects the glucose environment in which the erythrocyte circulates. Hemoglobin A.sub.1 is a descriptive term which describes all the fast hemoglobins which include HbA.sub.1c as well as HbA.sub.1a and HbA.sub.1b. Because many of these hemoglobins have glucose or glucose breakdown products (phosphorylated glycolytic intermediates) attached, they are referred to as glycosylated hemoglobins and also reflect average glucose over time; however, the value for glycosylated hemoglobins is about 50% higher than the measurement of HbA.sub.1c (or glucosylated hemoglobin) alone.
Table 4 summarizes the available clinical methods of measurement for circulating glycated proteins and the relative benefits and disadvantages of each method. Today a number of HbA.sub.1c column methods are available in addition to new radioimmunoassay methods, isoelectric focusing methods, and calorimetric methods. This later measurement can be performed on a spot of capillary blood placed on a filter paper shipped to a central laboratory. One can obtain a glycohemoglobin or glycated serum protein measurement by sending the sample to any one of a number of commercial laboratories. Fructosamine assays of serum proteins have become popular because they are amenable to automation; however, they have the major disadvantage of being influenced by ingested oxidants and reductants thus leading to considerable intraindividual variation not related to glycemia.
TABLE 4 ______________________________________ Classification of Current Clinical Assays ______________________________________ I. Physical methods based on changes in p1 A. Cation exchange chromatography PRO: Inexpensive and rapid CON: Sensitive to small changes in resin packing, ionic strength, pH, temperature, column loading, and affected by the labile fraction. Variant hemoglobins may interfere. B. High performance liquid chromatography (HPLC) PRO: Dedicated instruments avoid many problems in 1 CON. Relatively expensive C. Agarose gel eletrophoresis PRO: Inexpensive, minimal technician time, standardized plates and conditions in kits less sensitive to pH, triglyceride concentrations, and temperature CON: Precision problems induced by scanner and loading variation; sensitive to liabile fraction and variant hemoglobin. D. Isoelectric focusing PRO: Separates most minor hemoglobin variants CON: Precision over time dependent on use of same batch of ampholines on standardized plates; scanning effects precision II. Methods based on chemical principles A. Thiobarbituric acid/colorimetric assay PRO: Minimally effected by storage condition, fructose or 5-hydroxy-methyl furfural standards may be incorporated, filter paper assays available CON: Difficult to establish, large amount of technical time required, and affected by labile fraction B. Affinity chromatography with immobilized m-phenyl- boronate PRO: Rapid, inexpensive, minimally effected by chromatographic conditions, eliminates labile adduct CON: Resins vary within and between manufacturers C. Fructosamine determination by nitroblue tetrazolium reduction PRO: Inexpensive, standards incorporated, may be automated, not effected by labile adduct CON: Only for serum, lipids may interfere, reducing substances (in diet) may interfere III. Immunoassay PRO: Inexpensive, rapid, sensitive, specific, not effected by labile adduct CON: Antibodies difficult to raise ______________________________________
As noted above, in 1912, Louis Camille Maillard at the Sorbonne reported that aqueous solutions of reducing sugars turned progressively yellow-brown when heated or when stored under physiological conditions. Maillard LC: Reaction generale des acides amines sur les sucres; ses consequences biologiques. C.R. Acad Sci 1912; 154:66-68. For the next 60 years progress in understanding Maillard's reaction was largely restricted to the food industry. These fluorescent compounds were found to influence the flavor, taste, consistency, and overall appeal of foods as well as their nutritive properties. Kaanane A and Lubuza TP The Maillard Reaction in foods. In Baynes JW and Monnier VM (1989)The Maillard Reaction in Aging, Diabetes, and Nutrition. Alan R. Liss, New York, pp 301-328.
These fluorescent species can be found in vivo as well although a chemical assignment for the species involved is generally lacking. Evidence for the occurrence of the advanced Maillard reaction in long-lived molecules has been based in part on the presence of fluorescence that can be duplicated by incubation of proteins with glucose. Monnier VM, Kohm RR, Cerami A (1984). Accelerated age-related browning of human collagen in diabetes mellitus. Proc Natl Acad USA 81:853-857. Monnier VM, Vishwanath B, Frank KE, Elmets CA, Dauchot P, Kohn RR (1986). Relation between complications of Type I diabetes mellitus and collagen-linked fluorescence. N Engl J Med 314: 403408. Borohydride reducible as well as fluorescent products that co-chromatograph with similar molecules isolated from glucose-incubated proteins have been detected in lens and collagen. Monnier VM, Cerami A (1983). Nonenzymatic glycosylation and browning of proteins in vivo. In The Maillard Reaction in Foods and Nutrition, Waller GR and Feather MS Eds, American Chemical Society, Symposium Series 215:431-449. Oimomi M, Maeda Y, Hata F, Kitamura Y, Matsumoto S, Baba S, lga T, Yamamoto M (1988). Glycation of cataractous lens in nondiabetic senile subjects and in diabetic patients. Exp Eye Res 46:415-420. Further studies on the nature of the fluorophores that accumulate in aging human collagen revealed the presence of two major fluorophores with excitation-emission maxima at 328/378 nm, 335/385 nm and 360/460 nm respectively. McCance DR, Dyer DB, Dunn JA, Bailie KE, Thorpe SR, Baynes JW, Lyons TJ. Maillard reaction products and their relation to complications in insulin-dependent diabetes mellitus. J. Clin. Invest. 91: 2470-2478, 1993. Monnier VM. Toward a Maillard Reaction theory of aging. In Baynes JW and Monnier VM (1989) The Maillard Reaction in Aging, Diabetes, and Nutrition. Alan R. Liss, New York, pp1-21. Dyer DG, Dunn JA, Thorpe SR, Bailie KE, Lyons TJ, McCance DR, Baynes JW. Accumulation of Maillard reaction products in skin collagen in diabetes and aging. Journal of Clinical Investigation, 1993 Jun. 91(6) :2463-9. 3-deoxyglucosone, an intermediate product of the Maillard reaction with an excitation wavelength of 370nm and an emission of 440 nm has also been characterized. Kato H, Hayase F, Shin DB, Oimomi M, Baba S. 3-Deoxyglucosone, an intermediate product of the Maillard Reaction. In Baynes JW and Monnier VM (1989)The Maillard Reaction in Aging, Diabetes, and Nutrition. Alan R. Liss, New York, pp69-84. Albumin has also been shown to form fluorescent products following glucose incubation with an excitation maxima at 278 nm and emission at 340 nm. Suarez G. Nonenzymatic browning of proteins and the sorbitol pathway. In Baynes JW and Monnier VM (1989) The Maillard Reaction in Aging, Diabetes, and Nutrition. Alan R. Liss, New York, pp141-162. Non tryptophan fluorescence development with excitation and emission maxima at 330nm and 405 nm respectively have been shown to occur with both glucose and fructose (Walton DJ, McPherson JD, Shilton BH. Fructose mediated crosslinking of proteins. In Baynes JW and Monnier VM (1989) The Maillard Reaction in Aging, Diabetes, and Nutrition. Alan R. Liss, New York, pp163-170.) and these fluorescent species have been shown to bind rather tightly to albumin and IgG thus being present in blood. Suarez G. Nonenzymatic browning of proteins and the sorbitol pathway. In Baynes JW and Monnier VM (1989) The Maillard Reaction in Aging, Diabetes, and Nutrition. Alan R. Liss, New York, pp141-162.
Diabetic patients were found to have significantly elevated levels of serum peptide and hemoglobin advanced glycosylation end products determined by radioreceptor assay and a competitive ELISA format. Makita Z, Radoff S, Rayfield EJ, Yang Z, Skolnik E, Delaney V, Friedman EA, Cerami A, Vlassara H. Advances glycosylation end products in patients with diabetic nephropathy. (1991) N Eng J of Med 325: 836-842. Makita Z, Vlassara H, Rayfield E, Cartwright K, Fridman E, Rodby R, Cerami A, Bucala R. Hemoglobin-AGE: A circulating marker of advanced glycosylation. (1992). Science 258:651-653. This latter method has also been used to show increased levels of advanced glycosylation end products on hemoglobin from diabetic humans compared to nondiabetic subjects and the values correlated with glycated hemoglobin values as quantified by HbAlc determination by high performance cation exchange liquid chromatography. Makita Z, Radoff S, Rayfield EJ, Yang Z, Skolnik E, Delaney V, Friedman EA, Cerami A, Vlassara H. Advances glycosylation end products in patients with diabetic nephropathy. (1991) N Eng J of Med 325:836-842. Streptozotocin treated diabetic rats have been shown to have ELISA detected increases in advanced glycosylation end products in the urine when compared to controls. Koenig RJ, Peterson CM, Kilo C, Cerami A, Williamson JR: Hemoglobin A.sub.1c as an indicator of the degree of glucose intolerance in diabetes. Diabetes 1976; 25:230-232. As noted, several investigators have found that advanced Maillard reaction products accumulate in skin collagen. McCance DR, Dyer DB, Dunn JA, Bailie KE, Thorpe SR, Baynes JW, Lyons TJ. Maillard reaction products and their relation to complications in insulin-dependent diabetes mellitus. J. Clin. Invest. 91: 2470-2478, 1993. Monnier VM. Toward a Maillard Reaction theory of aging. In Baynes JW and Monnier VM (1989) The Maillard Reaction in Aging, Diabetes, and Nutrition. Alan R. Liss, New York, pp1-21. Dyer DG, Dunn JA, Thorpe SR, Bailie KE, Lyons TJ, McCance DR, Baynes JW. Accumulation of Maillard reaction products in skin collagen in diabetes and aging, Journal of Clinical Investigation, 1993 Jun, 91 (6): 2463-9. All of the above methodologies require sampling of a biologics tissue or fluid; however, they do confirm the presence of late Maillard reaction products in plasma, red cells, and tissues.